1. Programmed Cell Death A genetically controlled cell suicide pathway

2. video

3. The Morphology of Apoptosis
Engulfment of the cell corpse
Chromosomes condense and fragment
Nuclear membrane breaks down
Apoptotic body formation
Cytoplasm shrinks

4. Difference Between Apoptosis and Necrosis
Necrosis (pathological cell death): dying cells swell and lyse; toxic contents leak out and result in inflammatory response.
Apoptosis (physiological or programmed cell death): dying cells shrink, are engulfed and degraded by other cells, leave no trace, and don’t result in harmful outcomes

5. Functions of apoptosis
Sculpt body structures, e.g. hand digit
Serve some function but no longer needed
e.g. tadpole tail of frog.
Needed in one sex but not another
e.g. Mullerian duct important for female
is eliminated in males by apoptosis.
Produced in excess, e.g. extra neurons are
removed by apoptosis during neurogenesis.
Serve in immune system as a defense
mechanism to get rid of harmful or
damaged cells.

6. The Nematode C. elegans As a Model Organism in the Study of PCD
A great genetic system
Completely defined cell lineage
Study of cell death at a single cell resolution in living animals

7. The C. elegans Cell Lineagez y g e t o B A S M E C D 4 P

8. Cell Death Can Be Studied at a Single Cell Resolution
P11 X X
P11aap
Adapted from Sulston and Horvitz, Dev. Bology 56, , 1977

9. The First Cell Death Mutants Identified in C. elegans
In 1976, J. Sulston first described programmed cell death in nematodes and reported the first cell death mutant (nuc-1), in which DNA in the death cells fail to be degraded.
Wild-type
nuc-1

11. What went wrong with the ced-1 mutant?
In 1980, E. Hedgecock isolated two cell death mutants
(ced-1 and ced-2) which are pivotal for identification
of the other cell death genes.
Wild-type
ced-1
What went wrong with the ced-1 mutant?

12. What could go wrong with the ced-1 mutant?
a) Apoptotic cells fail to die
b) Normal cells die ectopically
c) Apoptotic cells fail to be engulfed
d) Normal cells are ectopically engulfed
e) Cells undergo necrosis
a and b
b and c
c and d
d and e
e and a
ced-1
Wild-type

13. What is next? Phenotypic analysis of ced-1 and ced-2 mutants
More cell deaths?
Dying cells cannot be removed or engulfed
How to distinguish these two possibilities?
Follow the cell lineage in the mutant animals
What is next?

14. Suppressor screens: ced-3 and ced-4
What are the functions of ced-3 and ced-4?
H. Ellis and R.H. Horvitz

15. What could be the functions of ced-3 and ced-4?
ced-3 and ced-4 promote cell corpse engulfment
2) Inhibitors of cell corpse engulfment
3) ced-3 and ced-4 could promote cell deaths
4) ced-3 and ced-4 could inhibit cell death
5) ced-3 and ced-4 could promote necrosis
1 and 2
2 and 3
3 and 4
4 and 5
5 and 1

16. What are the functions of ced-3 and ced-4?
ced-3 and ced-4 promote cell corpse engulfment?
Then the mutations must be increase-of-function
2) Inhibitors of cell corpse engulfment?
Then the mutations should be loss-of-function
3) ced-3 and ced-4 could promote cell deaths
How to distinguish 2) and 3)?

17. How do ced-3 and ced-4 kill the cells? Cells die by murder?
Lineage analysis suggest:
Many cells that normally die now survive
ced-3 and ced-4 are involved in cell killing
How do ced-3 and ced-4 kill the cells?
Cells die by murder?
Cells die by suicide?
cells die by aging?
Cells die because of injuries?
Cells die by sickness?

18. Mosaic Analysis
Using specific techniques to create genetically mosaic worms, in which some cells carry a wild-type gene and others are homozygous mutant. Analysis of such animals can reveal where in the animal a gene is needed for its functions
How to generate C. elegans genetic mosaics?
Mosaic animals have been generated by the spontaneous mitotic loss of an extrachromosomal genetic element that carries the wild-type allele of a gene in an otherwise homozygous mutant background.
free chromosome fragments
extrachromosomal arrays

19. unc-36 mosaic analysis
UNC-36 acts in ABp cell lineage

20. Cells Die by Suicide Rather Than Murder
Yuan and Horvitz demonstrated by mosaic analysis that ced-3 and ced-4 function in the dying cells to kill.
ced-3 encodes a protein with homology with IL-1b converting enzyme (ICE), a cysteine protease.
ced-4 encodes a protein similar to apoptotic protease-activating factor (Apaf-1).

21. Caspases Are Cell Death Executors
Yuan’s group using cell culture experiments showed ICE and CED-3 can both kill in mammalian cells
CED-3/ICE define a family of cysteine proteases, named caspases (aspartate-specific proteases), which so far has at least 12 family members
A caspase is first synthesized as an inactive protease precursor and later activated by specific proteolysis at specific aspartate residues.

22. Caspase Family caspase
Adapted from Thornberryand Lazebnik, Science 281, , 1998

23. Stucture of Caspase-3 (CPP32)
Adapted from Thornberryand Lazebnik, Science 281, , 1998

24. Activation of Caspases
1) By self-activation
2) By another cysteine protease or caspase

25. cps-6
ceh-30
CEM cells
psr-1
wah-1